The overall goal of my project is to develop and test methods to target proteins for characterization whose sequences are intermediate between a pair of related proteins with divergent structural and functional properties. In particular, interest falls in determining structural variation within the medically relevant class of salivary lipocalins. These proteins are found in the saliva of several bloodsucking insects, which are known vectors of Chagas1 disease. Identification of triabin, a related protein with similar biological function but a different topology, has prompted investigation into the mechanism which underlies its unique structure. To this end we have identified three sequence intermediate lipocalins which link triabin to nitrophorin 2, a related lipocalin with known structure and function. The goal of the proposed research is to develop protocols for cloning, expressing, and purifying these sequence intermediate lipocalins. The second aim of my research will be to determine the structure of at least one of them by NMR and x-ray crystallographic techniques. One histidine-tagged sequence intermediate has been cloned, expressed, refolded, and purified, and has proven itself amenable to studying by NMR. The other two proteins are in different stages of expression, refolding, and purification. They will also be brought along for structural studies, but at a lower priority. Additionally, I will develop a protocol to purify and refold untagged variants. Comparison of 2D correlation (HSQC) spectra between the tagged and the untagged variants will be critical for assessing whether the tagged forms display structural propertied consistent with a native structure. The likely outcome from this research will be structural determination of a sequence intermediate salivary lipocalin. With this structure, we will be able to more accurately determine the sequence requirements that contribute to the structural differences seen between triabin and nitrophorin 2, and understand some of the structural variation seen within this family. Understanding protein structural and functional variation is important in designing therapeutic targets, determining how parasites interact with hosts, and identifying proteins which have the potential to form into disease states. We are developing and assessing a method of target identification of proteins whose sequences are intermediate between those of known divergent properties. The overarching goal of this research is to increase our ability to annotate gene products with structural and functional information.